FIELD OF THE INVENTION
[0001] The invention relates to an antibacterial protein (bacteriocin) for the control of
Pseudomonas, preferably of
Pseudomonas aeruginosa, and to a nucleic acid molecule encoding the bacteriocin. The invention also relates
to a composition, notably a pharmaceutical composition, comprising the bacteriocin
or a combination of the bacteriocins. The invention further relates to the bacteriocin
or composition for use in therapy, preferably for the treatment of lung infection
or keratitis. The invention further relates to a method of treating or preventing
bacterial infection in a mammal, preferably in a human.
BACKGROUND OF THE INVENTION
[0002] Pseudomonas aeruginosa (P. aeruginosa) are opportunistic Gram negative pathogenic bacteria, causing both acute and chronic
infections. They harbor in their genome a large arsenal of virulence factors and antibiotic
resistance determinants, conferring remarkable metabolic flexibility and the ability
to adapt to multiple conditions, including the host immune response
1. Furthermore, rapid development of resistance to previously effective antimicrobials,
such as fluoroquinolones, aminoglycosides, and polymyxins, has been observed
2. This is of particular concern for conditions due to the biofilm colonization of
lungs in cystic fibrosis patients, urinary tract infections, and ear and eye infections.
The involvement of
P.
aeruginosa in a wide range of biofilm-related infections often leads to treatment failures
3. Thus, new antimicrobial substances are urgently needed.
[0003] The possibility to use bacteriocins as new generation antimicrobials has been suggested
previously
4.
P. aeruginosa produce colicin-type bacteriocins, pyocins, belonging to different classes: deoxyribonucleases,
ribonucleases, pore-forming proteins, peptidoglycan synthesis-blocking proteins, lectin-like
proteins, and bacteriophage tail-like protein complexes
5,6. Several studies have described the use of natural pyocins for successful treatment
of
P.
aeruginosa infections in various animal models
7-12.
[0004] The main hurdles in the clinical development of bacteriocin antimicrobials are the
proteinaceous nature of bacteriocins and their limited activity spectrum. Being proteins,
colicin type bacteriocins are expected to present the same challenges observed in
the clinical development of bacteriophage lysins when used intravenously: short half-life,
immunogenicity and weakened activity in serum
13,14. These problems might be less relevant in the development of topical, oral or inhaled
antimicrobials.
[0005] Previously, we successfully expressed in a plant transient expression system six
different pyocins
11. The only so far known
P. aeruginosa pore-forming pyocin, S5, was found to be active against 40% of tested clinical isolates.
S5 demonstrated superior activity compared to all other pyocins in reducing bacterial
numbers in liquid culture and biofilm assays and also was most efficacious in protecting
G. mellonella larvae from death due to
P. aeruginosa infection
11.
[0006] Self-inhibition of S5-producing strains was prevented by co-expression of an immunity
gene, located downstream of the bacteriocin gene. Lethality due to pore formation
by pyocin S5 was transiently impeded along the secretory route via a membrane-integrated
immunity protein consisting of three transmembrane helices (TMHs)
15.
[0007] A general problem with bacteriocins in their limited breadth of activity or, in other
words, quite high specificity against target strains or species of
Pseudomonas, or against strains of
P. aeruginosa, which is problematic if the pathogenic agent that causes an infection is not known
or has not been identified, yet.
[0008] Therefore, it is an object of the present invention to provide bacteriocins active
against
Pseudomonas for the control of
Pseudomonas. Further, it is an object to provide bacteriocins having a broad activity range against
multiple strains of
Pseudomonas. It is a further object to provide a remedy against infection by
Pseudomonas, notably by
P.
aeruginosa.
SUMMARY OF THE INVENTION
[0009] These objects are accomplished by:
- 1) A (chimeric) antibacterial protein comprising a polypeptide comprising a first
polypeptide segment and, preferably contiguous thereto, a second polypeptide segment,
said first polypeptide segment being selected from the following group (a-i) to (d-i),
said second polypeptide segment being selected from the following group (a-i)' to
(d-iii)':
group (a-i) to (d-i):
(a-i) the segment of the amino acid sequence of SEQ ID NO: 1 (translocation + receptor binding domain of S5), or
(b-i) a segment having at least 80%, more preferably at least 85%, even more preferably
at least 90%, and even more preferably at least 95% sequence identity to the amino
acid sequence of SEQ ID NO: 1, or
(c-i) a segment having at least 85%, preferably at least 90%, more preferably at least
95%, and most preferably at least 98 % sequence similarity to the amino acid sequence
of SEQ ID NO: 1, or
(d-i) a segment having from 1 to 60, preferably from 1 to 45, more preferably from
1 to 30, and most preferably from 1 to 15 amino acid residue substitutions, additions,
insertions and/or deletions compared to the amino acid sequence of SEQ ID NO: 1;
group (a-i)' to (d-iii)':
(a-i)' the segment of the amino acid sequence of SEQ ID NO: 3 (cytotoxicity domain of PmnH), or
(a-ii)' the segment of the amino acid sequence of SEQ ID NO: 7 (cytotoxicity domain of Pflu095), or
(a-iii)' the segment of the amino acid sequence of SEQ ID NO: 11 (cytotoxicity domain of Pflu373); or
(b-i)' a segment having at least 80%, more preferably at least 85%, even more preferably
at least 90%, and most preferably at least 95% sequence identity to the amino acid
sequence of SEQ ID NO: 3, or
(b-ii)' a segment having at least 80%, preferably at least 85%, more preferably at
least 90%, and most preferably at least 95% sequence identity to the amino acid sequence
of SEQ ID NO:7, or
(b-iii)' a segment having at least 80%, preferably at least 85%, more preferably at
least 90%, and most preferably at least 95% sequence identity to the amino acid sequence
of SEQ ID NO:11; or
(c-i)' a segment having at least 85%, preferably at least 90%, and more preferably
at least 95%, and most preferably at most 98% sequence similarity to the amino acid
sequence of SEQ ID NO: 3, or
(c-ii)' a segment having at least 85%, preferably at least 90%, and more preferably
at least 95%, and most preferably at most 98% sequence similarity to the amino acid
sequence of SEQ ID NO: SEQ ID NO: 7, or
(c-iii)' a segment having at least 85%, preferably at least 90%, and more preferably
at least 95%, and most preferably at most 98% sequence similarity to the amino acid
sequence of SEQ ID NO: SEQ ID NO: 11; or
(d-i)' a segment having from 1 to 38, preferably from 1 to 28, more preferably from
1 to 19, and most preferably from 1 to 10 amino acid residue substitutions, additions,
insertions and/or deletions compared to the amino acid sequence of SEQ ID NO: 3, or
(d-ii)' a a segment having from 1 to 38, preferably from 1 to 28, more preferably
from 1 to 19, and most preferably from 1 to 10 amino acid residue substitutions, additions,
insertions and/or deletions compared to the amino acid sequence of SEQ ID NO: 7, or,
(d-iii)' a segment having from 1 to 38, preferably from 1 to 28, more preferably from
1 to 19, and most preferably from 1 to 10 amino acid residue substitutions, additions,
insertions and/or deletions compared to the amino acid sequence of SEQ ID NO: 11.
- 2) A (chimeric) antibacterial protein comprising a polypeptide comprising or consisting
of:
(A-i) the amino acid sequence of SEQ ID NO: 5 (sequence of entire protein S5-PmnH), or
(A-ii) the amino acid sequence of SEQ ID NO: 9 (sequence of entire protein S5-Pflu095), or
(A-iii) the amino acid sequence of SEQ ID NO: 13 (sequence of entire protein S5-Pflu373); or
(B-i) an amino acid sequence having at least 80%, more preferably at least 85%, even
more preferably at least 90%, and even more preferably at least 95% sequence identity
to the amino acid sequence of SEQ ID NO: 5, or
(B-ii) an amino acid sequence having at least 80%, more preferably at least 85%, even
more preferably at least 90%, and even more preferably at least 95% sequence identity
to the amino acid sequence of SEQ ID NO: 9, or
(B-iii) an amino acid sequence having at least 80%, more preferably at least 85%,
even more preferably at least 90%, and even more preferably at least 95% sequence
identity to the amino acid sequence of SEQ ID NO: 13; or
(C-i) an amino acid sequence having at least 85%, preferably at least 90%, and more
preferably at least 95%, and most preferably at least 98% sequence similarity to the
amino acid sequence of SEQ ID NO: 5, or
(C-ii) an amino acid sequence having at least 85%, preferably at least 90%, and more
preferably at least 95%, and most preferably at least 98% sequence similarity to the
amino acid sequence of SEQ ID NO: 9, or
(C-iii) an amino acid sequence having at least 85%, preferably at least 90%, and more
preferably at least 95%, and most preferably at least 98% sequence similarity to the
amino acid sequence of SEQ ID NO: 13; or
(D-i) an amino acid sequence having from 1 to 100, preferably from 1 to 75, more preferably
from 1 to 50, even more preferably from 1 to 25, and most preferably from 1 to 12
amino acid residue substitutions, additions, insertions and/or deletions to the amino
acid sequence of SEQ ID NO: 5, or
(D-ii) an amino acid sequence having from 1 to 100, preferably from 1 to 75, more
preferably from 1 to 50, even more preferably from 1 to 25, and most preferably from
1 to 12 amino acid residue substitutions, additions, insertions and/or deletions to
the amino acid sequence of SEQ ID NO: 9; or
(D-iii) an amino acid sequence having from 1 to 100, preferably from 1 to 75, more
preferably from 1 to 50, even more preferably from 1 to 25, and most preferably from
1 to 12 amino acid residue substitutions, additions, insertions and/or deletions to
the amino acid sequence of SEQ ID NO: 13.
- 3) The antibacterial protein according to item 1, wherein said first segment is N-terminal
to said second segment in the amino acid sequence of said antibiotic protein.
- 4) A composition comprising a protein according to any one of items 1 to 3.
- 5) The composition according to item 4, further comprising a second bacteriocin according
to any one of items 1 to 3.
- 6) A pharmaceutical composition comprising an antibacterial protein as defined in
any one of items 1 to 3 or comprising a composition according to any one of items
4 and 5, and one or more pharmaceutically acceptable carrier(s) or excipient(s).
- 7) The pharmaceutical composition according to item 6, which is a sterile aqueous
solution comprising said antibacterial protein or is a solid formulation, preferably
said solid formulation is a powder suitable for reconstitution in an aqueous liquid
medium or is a solid formulation suitable for administration as an aerosol.
- 8) The antibacterial protein according to any one of items 1 to 3, or a composition
according to any one of items 4 to 7 for use in therapy.
- 9) The antibacterial protein according to any one of items 1 to 3, or a composition
according to any one of items 4 to 7, for use in the treatment or prevention of a
bacterial infection, preferably of a bacterial infection by Pseudomonas, more preferably by P. aeruginosa.
- 10) The antibacterial protein according to any one of items 1 to 3, or a composition
according to any one of items 4 to 7, for use in therapy, preferably for the treatment
of lung infection or keratitis.
- 11) The antibacterial protein according to any one of items 1 to 3, or a composition
according to any one of items 4 to 7, for use in the treatment of lung infection by
administration into the lungs of a mammal as a solid or liquid aerosol; or for the
treatment of keratitis by administration into an affected eye of a mammal in the form
of an aqueous solution.
- 12) A method of treating or preventing bacterial infection in a mammal, comprising
administering to a mammal in need thereof an antibacterial protein as defined in any
one of items 1 to 3, or a composition according to any one of items 4 to 6, or a pharmaceutical
composition according to item 7.
- 13) The method according to item 12, wherein bacterial lung infection is treated by
administration into the lungs of a mammal as a solid or liquid aerosol; or wherein
bacterial keratitis is treated by administration into an affected eye of a mammal
in the form of an aqueous solution.
- 14) A nucleic acid molecule comprising a nucleic acid sequence encoding a protein
according to any one of items 1 to 3.
- 15) The nucleic acid molecule according to item 14, said nucleic acid sequence comprising
a polynucleotide according to any one of SEQ ID NOs: 6, 10, or 14.
- 16) Bacterial or eukaryotic cell comprising a nucleic acid molecule according to item
14 or 15.
[0010] The inventors have considered ways of broadening the spectrum of activity of anti-
Pseudomonas bacteriocins, such as pyocin S5. The inventors have surprisingly found that the spectrum
of activity of pyocin S5 can be broadened by modifying its killing domain to avoid
its recognition by the immunity protein of the target pathogen. Other
Pseudomonas species (in particular
P. fluorescens) harbour in their genomes a variety of genes coding for pore forming bacteriocin-like
proteins
6,16. Through the mining of GenBank database, the inventors retrieved several sequences
of putative pore-forming bacteriocins from different
Pseudomonas species and selected six colicin E1-like and colicin A-like proteins. The inventors
then used the cytotoxic pore forming domains of these bacteriocins to construct chimeric
proteins fusing them to the receptor-binding and translocation domains of pyocin S5.
The inventors tested the activity of the resulting chimeric bacteriocins
in vitro and, surprisingly, could identify bacteriocins with high cytotoxic activity and broadened
specificity against
Pseudomonas strains. The inventors found that the identified chimeric bacteriocins are active
in different animal disease models,
P. aeruginosa keratitis and lung colonization murine models, using topical and inhaled administration
of the chimeric bacteriocins of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0011]
Fig. 1 Sequence analysis of pore forming domains (pfam01024) of putative Pseudomonas bacteriocins. A. ClustalW amino acid sequence alignment of Pseudomonas bacteriocins with pyocin S5. The sequences shown, from top to bottom, are those of
SEQ ID NOs: 3, 19, 23, 15, 11, 7, and 39, respectively. B. Neighbor-Joining tree alignment of known E. coli, Klebsiella bacteriocins, pyocin S5, PmnH and putative P. fluorescens and P. putida bacteriocins. Col28B (CAA44310.1), ColE1 (AAA87379.1), Col10 (CAA57998.1), Col5 (CAA61102.1),
Collb (AAA23188.1), Colla (WP_001283344.1), CoIN (P08083.1), ColA (P04480.1), ColU
(CAA72509.1), ColB (P05819.3), ColY (AAF82683.1), KpneA (SAV78255.1), Kvarla (KDL88409),
Pyocin S5 (WP_003115311), PmnH (EIK72868.1), Pflu618 (WP_034155618), Ppu259 (WP_098964259),
Pflu794 (WP_081041794), Pflu373 (WP_014717373), Pflu095 (WP_016979095).
Fig. 2 SDS-PAGE Coomassie staining of purified chimeric pyocins. Lane 1 - PageRuler Prestained
protein ladder (Thermo Fisher Scientific), lane 2 - S5 (0.9 mg/ml), lane 3 - S5-PmnH
(0.9 mg/ml), lane 4 - S5-Pflu095 (0.9 mg/ml), lane 5 - S5-Pflu373 (0.9 mg/ml), lane
6 - S5-Pflu794 (0.9 mg/ml), lane 7 - S5-Pflu618 (0.9 mg/ml), Lane 8 - S5-Pput259 (1.4
mg/ml). 4 µl of proteins per lane.
Fig. 3 Pyocin S5 and chimeric pyocin activities on pyocin S5-producing P. aeruginosa strains. 0.3, 3 and 30 µg of S5 and chimeric bacteriocins were spotted on 6 mm Whatman discs placed on CAA agar lawns
of different P. aeruginosa strains and incubated overnight.
Fig. 4 CFU counts in ex vivo porcine corneas, infected with P. aeruginosa ATCC 19660 or
PAO1 and treated with S5-PmnH. 3x104 CFU of P. aeruginosa ATCC 19660 strain or 0.4x104
CFU of P. aeruginosa PAO1 strain were applied to cornea and incubated for 16-20 hours
at 37 °C. Then 5 µg of S5-PmnH were applied to cornea and incubated for additional
16-20 hours. Statistical significances of the quantitative data were analyzed using
GraphPad Prism software by the 1-way repeated measures ANOVA and Bonferroni's' correction
for multiple comparisons. Mean is indicated by horizontal bar. ***P≤0.001, ****P≤0.0001 vs vehicle-treated mice.
Fig. 5 Mice cornea infection by cytotoxic strain ATCC 19660 and treatment by S5-PmnH or
tobramycin. A. CFU counts and clinical scores of mice eyes, when treatment started 30 min post-infection.
B - CFU counts and clinical scores of mice eyes, when treatment started 6 h post infection.
C. Hematoxylin-eosin staining of cornea sections. Left panel - treatment with pyocin started 30 min post-infection. Uninfected eyes - no observed
microscopic aberations. Infected control eyes: day 1 - strong corneal inflamation,
days 3 and 5 - the histology was not possible because of disrupted structure of the
eye. Weak edema in the corneal stroma is observed in all infected eyes treated with
S5-PmnH and with tobramycin. Right panel - treatment with pyocin started 6 h post infection. Uninfected eyes: no marked cornea
abberations. Infected untreated eyes: day 1 - thinning of corneal epithelium, thickening
of stroma, acute inflammation, days 3 and 5 - acute suppurated inflammation of cornea.
S5-PmnH-treated infected eyes: day 1 - acute inflammation of cornea, days 3 and 5
- no marked aberrations, week edema of corneal stroma. Tobramycin-treated infected
eyes: day 3 and 5 - slight thickening of epithelium. Statistical significances of
the quantitative data were analyzed using GraphPad Prism software by the 2-way repeated
measures ANOVA and Dunnett's correction for multiple comparisons. Mean is indicated
by horizontal bar. ***P≤0.001, ****P≤0.0001 vs vehicle-treated mice.
Fig. 6 Mice cornea infection by invasive strain PAO1 and treatment by S5-PmnH or tobramycin.
The corneas of left eye of mice were infected with 4×106 CFU of P. aeruginosa PAO1 strain. The treatment with 20 µg of S5-PmnH or 140 µg of tobramycin started
6 h post-infection and was applied twice daily. A. CFU counts in mice corneas at 1, 3 and 5 dpi. B. Cornea clinical scores at day 1, day 3 and day 5 of experiment. C. Hematoxylin-eosin staining of cornea sections. Uninfected eyes: no marked aberrations,
weak edema in corneal stroma observed in most samples. Infected S5-PmnH-treated eyes:
days 1 and 3 - acute inflammation of cornea, day 5 - no aberrations. Infected tobramycin-treated
eyes: Day 1 - acute inflammation of cornea, local cornea lesions, Day 3 - thinning
of cornea epithelium, strong edema of the corneal stroma; day 5 - local inflammation
of cornea, thinning and degeneration of cornea epithelium. Statistical significances
of the quantitative data were analyzed using GraphPad Prism software by the 2-way
repeated measures ANOVA and Dunnett's correction for multiple comparisons. Mean is
indicated by horizontal bar. **P≤0.01, ***P≤0.001, ****P≤0.0001 vs vehicle-treated mice. Not significant (ns) P>0.05.
Fig. 7. Scatterplot of terminal lung burden following IN infection with P. aeruginosa ATCC 27853. The data from the culture burdens were analyzed using appropriate non-parametric
statistical models (Kruskal-Wallis using Conover-Inman to make all pairwise comparisons
between groups) with StatsDirect (v. 3.3.3). The geometric mean burden of each treatment
is indicated by the horizontal bar. *P ≤0.05, **P ≤ 0.0005, *** P ≤ 0.0001, compared
to vehicle control. LOD = limit of detection.
Fig. 8. PCR amplification of S5 killing and immunity genes and fptA from genomic DNA of
25 P. aeruginosa isolates. Sequence-specific primers were designed to amplify 1500 bp fragment of
S5K, 330 bp fragment of S5I and 556 bp fragment of fptA. FptA is amplified in all
tested strains, S5K and S5I was amplified in PA14, PAO1, HP6, HP7, ATCC 19660 and
NCTC 13921 strains. K - negative control.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The bacteriocin of the invention is a colicin-type antibiotic protein. Colicins,
in the case of
E. coli colicins, are plasmid-encoded cytotoxins synthesized by
Escherichia coli, which are secreted into the medium and kill sensitive strains of
E. coli. Colicins may belong to different cytotoxic classes according to the mechanism by
which they kill sensitive bacterial strains:
- pore-forming colicins such as ColA, CoIE1, CoIN, CoIK, Colla, Collb, and ColD, which
kill cells by causing membrane depolarization
- RNase colicins,
- DNase colicins, and
- inhibitors of cell wall synthesis such as ColM.
[0013] Colicins, as well as the bacteriocins of the invention comprise, along their amino
acid sequence, from the N-terminus to the C-terminus, three domains referred to as
receptor binding domain, translocation domain, and killing or cytotoxicity domain
(
Cascales et al., Colicin Biology, Microbiology and Molecular Biology Reviews, Vol.
71, (2007) 158-229). The receptor binding domain allows binding of the colicin to a receptor on the
outer membrane of a bacterial cell. The translocation domain allows binding to translocation
proteins of a target bacterial cell for translocation inside the target cell. The
bacteriocins of the invention are pore-forming bacteriocins. They exert cytotoxic
activity by pore formation in the inner cell membrane of target cells. Thus, their
target compartment of the target cells is the periplasm of the target cells. The translocation
domain of the bacteriocins of the invention allows translocation of the bacteriocins
into the periplasm of cells of a target pathogen.
[0014] The antibacterial protein of the invention (also referred to herein as "bacteriocin"
or "pyocin") is a pore-forming bacteriocin. It comprises a polypeptide comprising
a first polypeptide segment (briefly referred to herein as "first segment") and, generally
contiguous thereto, a second polypeptide segment (briefly referred to herein as "second
segment"). The first segment comprises the receptor binding domain and the translocation
domain (from the N-terminus to the C-terminus). The second segment comprises the pore
forming cytotoxic (or killing) domain. The bacteriocins of the invention are occasionally
referred to herein as "chimeric" or as "chimeric pyocins", as they comprises segments
that may be or are derived from different origins, notably from bacteriocins of different
species of
Pseudomonas.
[0015] Herein, a polypeptide segment (or, briefly, "segment") refers to a plurality of contiguous
amino acid residues of a polypeptide or protein, the polypeptide or protein having
a larger number of amino acid residues than the segment. Thus, a segment is a part
of a polypeptide or protein. The term "polypeptide" refers to a polypeptide molecule
(as opposed to a fragment, part or moiety of a molecule) without limitation with regard
to the number of amino acid residues of the polypeptide.
[0016] The term protein covers polypeptides and combinations or complexes of two or more
polypeptides, optionally with further components such as metal ions complexed by a
polypeptide. Generally, a protein may be monomeric, homo- or heterooligomeric (e.g.
dimeric), and may comprise further components such as metal ions complexed by one
or more polypeptide.
[0017] The term "amino acid sequence" refers to the sequence of amino acid residues of a
protein, polypeptide or segment; the term refers to the sequence information which
is structural information of a protein, polypeptide, or segment.
[0018] The first segment of the bacteriocin of the invention may be any one selected from
the following group (a-i) to (d-i):
(a-i) the segment of the amino acid sequence of SEQ ID NO: 1, or
(b-i) a segment having at least 80%, more preferably at least 85%, even more preferably
at least 90%, and even more preferably at least 95% sequence identity to the amino
acid sequence of SEQ ID NO: 1, or
(c-i) a segment having at least 85%, preferably at least 90%, more preferably at least
95%, and most preferably at least 98 % sequence similarity to the amino acid sequence
of SEQ ID NO: 1, or
(d-i) a segment having from 1 to 60, preferably from 1 to 45, more preferably from
1 to 30, and most preferably from 1 to 15 amino acid residue substitutions, additions,
insertions and/or deletions compared to the amino acid sequence of SEQ ID NO: 1.
[0019] The second segment of the bacteriocin of the invention may be any one selected from
the following group (a-i)' to (d-iii)':
(a-i)' the segment of the amino acid sequence of SEQ ID NO: 3 (killing domain of PmnH), or
(a-ii)' the segment of the amino acid sequence of SEQ ID NO: 7 (killing domain of Pflu095), or
(a-iii)' the segment of the amino acid sequence of SEQ ID NO: 11 (killing domain of Pflu373); or
(b-i)' a segment having at least 80%, more preferably at least 85%, even more preferably
at least 90%, and most preferably at least 95% sequence identity to the amino acid
sequence of SEQ ID NO: 3, or
(b-ii)' a segment having at least 80%, preferably at least 85%, more preferably at
least 90%, and most preferably at least 95% sequence identity to the amino acid sequence
of SEQ ID NO:7, or
(b-iii)' a segment having at least 80%, preferably at least 85%, more preferably at
least 90%, and most preferably at least 95% sequence identity to the amino acid sequence
of SEQ ID NO:11; or
(c-i)' a segment having at least 85%, preferably at least 90%, and more preferably
at least 95%, and most preferably at most 98% sequence similarity to the amino acid
sequence of SEQ ID NO: 3, or
(c-ii)' a segment having at least 85%, preferably at least 90%, and more preferably
at least 95%, and most preferably at most 98% sequence similarity to the amino acid
sequence of SEQ ID NO: SEQ ID NO: 7, or
(c-iii)' a segment having at least 85%, preferably at least 90%, and more preferably
at least 95%, and most preferably at most 98% sequence similarity to the amino acid
sequence of SEQ ID NO: SEQ ID NO: 11; or
(d-i)' a segment having from 1 to 38, preferably from 1 to 28, more preferably from
1 to 19, and most preferably from 1 to 10 amino acid residue substitutions, additions,
insertions and/or deletions compared to the amino acid sequence of SEQ ID NO: 3, or
(d-ii)' segment having from 1 to 38, preferably from 1 to 28, more preferably from
1 to 19, and most preferably from 1 to 10 amino acid residue substitutions, additions,
insertions and/or deletions compared to the amino acid sequence SEQ ID NO: 7, or
(d-iii)' a segment having from 1 to 38, preferably from 1 to 28, more preferably from
1 to 19, and most preferably from 1 to 10 amino acid residue substitutions, additions,
insertions and/or deletions compared to the amino acid sequence SEQ ID NO: 11.
[0020] The wording "the segment of the amino acid sequence of SEQ ID NO:z", z meaning any
one of the SEQ ID NOs referred to, means that the amino acid sequence of the segment
is that of SEQ ID NO:z, i.e. the entire amino acid sequence of SEQ ID NO:z. The wording
"a segment having at least ... sequence identity/similarity to the amino acid sequence
of SEQ ID NO: z" means that the amino acid sequence of the segment has at least the
indicated amino acid sequence identity or similarity, respectively, to the entire
amino acid sequence of SEQ ID NO: z, and preferably has at least the number of amino
acid residues as the SEQ ID NO referred to. The SEQ ID NOs with respect to which segments
or amino acid sequences are defined are generally referred to herein as reference
sequences.
[0022] Where a polypeptide or segment is defined herein by a number or number range of amino
acid residue substitutions, additions, insertions and/or deletions, amino acid residue
substitutions, additions, insertions or deletions may be combined, but the given number
or number or number range refers to the sum of all amino acid residue substitutions,
additions, insertions and deletions. Among amino acid residue substitutions, additions,
insertions and deletions, amino acid substitutions, additions, and deletions are preferred.
The term "insertions" relates to insertions of amino acid residues within the amino
acid sequence of a reference sequence, i.e. excluding additions at the C- or N-terminal
end of the reference sequence. The term additions means additions of amino acid residues
at the C- or N-terminal end of the amino acid sequence of a reference sequence. A
deletion may be a deletion of a terminal or an internal amino acid residue of a reference
sequence. Herein, where a polypeptide or segment is defined by a number or number
range of amino acid residue substitutions, additions, insertions and/or deletions
relative to a reference sequence, in a further embodiment, the polypeptide or segment
may have from 1 to several amino acid residue substitutions, additions, insertions
or deletions relative to the indicated amino acid sequence of segment.
[0023] In the bacteriocin of the invention, the first segment is preferably N-terminal to
said second segment in the amino acid sequence of said bacteriocin.
[0024] Herein, in any item (x-y)' (wherein x stands for any one of a, b, c, or d, and y
stands for any roman numeral i to iii), the prime ' indicates killing domains or segments.
Items (x-y) lacking the prime indicate the segments comprising the receptor binding
and translocation domains. Among items (a) to (d), those of items (a), (b) and (d)
are preferred and items (a) and (d) are more preferred. Similarly, among items (a)'
to (d)', those of items (a)', (b)' and (d)' are preferred and items (a)' and (d)'
are more preferred. This applies analogously to combinations of items (a) to (d) and
(a)' to (d)', where combinations of the same letter a to d are preferred.
[0025] Where the protein of the invention comprises a first segment as defined above and
a second segment as defined above, any first segment as defined above may be combined
with any second segment. In one embodiment, a binding and translocation segment of
any item (a) to (d) is combined with a catalytic domain or segment of any item (a)'
to (d)', respectively.
[0026] In a first general embodiment, a first segment of any one of items (a) to (d) is
combined with a second segment according to any one of items (x-i)', i.e. with a killing
domain of PmnH or a derivative thereof as defined in any one of items (x-i)'.
[0027] In a second general embodiment, a first segment of any one of items (a) to (d) is
combined with a second segment according to any one of items (x-ii)', i.e. with a
killing domain of Pflu095 or a derivative thereof as defined in any one of items (x-ii)'.
[0028] In a third general embodiment, a first segment of any one of items (a) to (d) is
combined with a second segment according to any one of items (x-iii)', i.e. with a
killing domain of Pflu373or a derivative thereof as defined in any one of items (x-iii)'.
[0029] The bacteriocin preferably has a minimum inhibitory concentration (MIC) as determined
by the assay of Example 2 against
P. aeruginosa PAO1 of at most 10 times, more preferably at most 5-times, even more preferably at
most 2.5 times of that of S5-Pflu095 of SEQ ID NO: 5.
[0030] The first and second segment may be or may not be linked by a peptide linker. Preferably,
no peptide linker is present. If a peptide linker is used, the peptide linker may
consist of from 1 to 50 amino acid residues, preferably from 1 to 30, and more preferably
from 1 to 10 amino acid residues. A peptide linker may be selected so as not to increase
its MIC in the assay of Example 2 against
P. aeruginosa PAO1, compared to a bacteriocin lacking the linker but having otherwise the same
amino acid sequence, by more than 5-fold, preferably not more than 2- fold. Alternatively,
the condition of the previous paragraph may be applied.
[0031] The bacteriocin of the invention may alternatively be defined as comprising a polypeptide
that comprises or consists of:
(A-i) the amino acid sequence of SEQ ID NO: 5 (sequence of protein S5-PmnH), or
(A-ii) the amino acid sequence of SEQ ID NO: 9 (sequence of protein S5-Pflu095), or
(A-iii) the amino acid sequence of SEQ ID NO: 13 (sequence of protein S5-Pflu373);
or
(B-i) an amino acid sequence having at least 80%, more preferably at least 85%, even
more preferably at least 90%, and even more preferably at least 95% sequence identity
to the amino acid sequence of SEQ ID NO: 5, or
(B-ii) an amino acid sequence having at least 80%, more preferably at least 85%, even
more preferably at least 90%, and even more preferably at least 95% sequence identity
to the amino acid sequence of SEQ ID NO: 9, or
(B-iii) an amino acid sequence having at least 80%, more preferably at least 85%,
even more preferably at least 90%, and even more preferably at least 95% sequence
identity to the amino acid sequence of SEQ ID NO: 13; or
(C-i) an amino acid sequence having at least 85%, preferably at least 90%, and more
preferably at least 95%, and most preferably at least 98% sequence similarity to the
amino acid sequence of SEQ ID NO: 5, or
(C-ii) an amino acid sequence having at least 85%, preferably at least 90%, and more
preferably at least 95%, and most preferably at least 98% sequence similarity to the
amino acid sequence of SEQ ID NO: 9, or
(C-iii) an amino acid sequence having at least 85%, preferably at least 90%, and more
preferably at least 95%, and most preferably at least 98% sequence similarity to the
amino acid sequence of SEQ ID NO: 13; or
(D-i) an amino acid sequence having from 1 to 100, preferably from 1 to 75, more preferably
from 1 to 50, even more preferably from 1 to 25, and most preferably from 1 to 12
amino acid residue substitutions, additions, insertions and/or deletions to the amino
acid sequence of SEQ ID NO: 5, or
(D-ii) an amino acid sequence having from 1 to 100, preferably from 1 to 75, more
preferably from 1 to 50, even more preferably from 1 to 25, and most preferably from
1 to 12 amino acid residue substitutions, additions, insertions and/or deletions to
the amino acid sequence of SEQ ID NO: 9; or
(D-iii) an amino acid sequence having from 1 to 100, preferably from 1 to 75, more
preferably from 1 to 50, even more preferably from 1 to 25, and most preferably from
1 to 12 amino acid residue substitutions, additions, insertions and/or deletions to
the amino acid sequence of SEQ ID NO: 13.
[0032] The wording "the amino acid sequence of SEQ ID NO: z" in items (A-y) means that the
amino acid sequence of the polypeptide is or comprises that of the reference sequence
(SEQ ID NO: z). The wording "an amino acid sequence having at least ... sequence identity/similarity
to the amino acid sequence of SEQ ID NO: z" of items (B-y) or (C-y), respectively,
means that the amino acid sequence of the polypeptide has at least the indicated amino
acid sequence identity or similarity, respectively, to the entire amino acid sequence
of SEQ ID NO: z, and preferably has at least the number of amino acid residues as
the SEQ ID NO referred to. Herein, the determination of sequence identities and similarities
is done as defined above.
[0033] The wording "an amino acid sequence having from ... amino acid residue substitutions,
additions, insertions and/or deletions to the amino acid sequence of SEQ ID NO: z"
(SEQ ID NO: z stands for the reference sequence) of items (D-y) means that the amino
acid sequence of the polypeptide has the indicated number or number range of amino
acid residue substitutions, additions, insertions and/or deletions to the entire amino
acid sequence of the reference sequence. Otherwise, the definitions and preferred
embodiments defined above apply analogously.
[0034] The bacteriocin of the invention may, in addition to the definition given above,
comprise a further segment at the N- and/or C-terminus, such as a purification tag,
signal sequence, etc. In one embodiment, however, the polypeptide of the bacteriocin
contains no further segment, but the polypeptide of the bacteriocin consists of the
definitions above.
[0035] The bacteriocin preferably has a minimum inhibitory concentration (MIC) as determined
by the assay of Example 2 against
P. aeruginosa PAO1 of at most 10 times, more preferably at most 5-times, even more preferably at
most 2.5 times of that of S5-Pflu095 of SEQ ID NO: 5.
[0036] The invention also provides a nucleic acid molecule and a nucleic acid construct,
comprising a nucleic acid sequence encoding a bacteriocin according to the invention.
The acid molecule and nucleic acid construct may comprise a transcription promoter
that is preferably active in the cells wherein the bacteriocin is to be expressed,
such as bacterial or plant cells.
[0037] The invention also provides a bacterial or eukaryotic cell, comprising the nucleic
acid molecule according to the invention. The eukaryotic cell may be a mammalian cell
or a plant cell. The plant cell may be a cell of a plant wherein the bacteriocin is
or is to be expressed. Further, a plant comprising the nucleic acid molecule according
to the invention is provided. The plant may be any one of those mentioned below.
[0038] The invention further provides a composition comprising a (first) bacteriocin of
the invention and optionally further components as the case requires such as one or
more carriers or excipients. The composition may further comprise a second or further
bacteriocin of the invention. Second bacteriocin means that it comprises a polypeptide,
the amino acid sequence of which differs from that of the first bacteriocin. Preferably,
the second bacteriocin has a killing domain from another origin than the first bacteriocin.
Among the bacteriocins of items (X-i) to (X-iii), X being selected from A, B, C, D,
this means that a bacteriocin of item (X-i) may, for example, be combined with a bacteriocin
of item (X-ii) or (X-iii).
[0039] As the bacteriocin of the invention may be produced by expression in plants or cells
thereof, the composition may be a plant material or extract of the plant material,
wherein the plant material may be a material from a plant having expressed the bacteriocin,
preferably
Nicotiana or an edible plant having expressed said bacteriocin. An extract of the plant material
may be an aqueous solution containing the bacteriocin of the invention that is present
or expressed in said plant material and optionally other water-soluble components,
or a dried product of such aqueous solution. The extract preferably has water-insoluble
components of the plant material removed e.g. by filtration or centrifugation. The
plant material may be a material from a plant selected from the group consisting of
spinach, chard, beetroot, carrot, sugar beet, leafy beet, amaranth,
Nicotiana, and/or said plant material may be one or more leaves, roots, tubers, or seeds, or
a crushed, milled or comminuted product of said leaves, roots, tubers, or seeds. In
a preferred embodiment, notably for use as a pharmaceutical composition, the bacteriocin
is purified to a level required by the respective regulatory provisions for the intended
application or use of the bacteriocin or composition of the invention.
[0040] The composition (such as said extract from a plant) may be a solid or liquid composition,
such as a solution or a dispersion, containing the bacteriocin(s) of the invention.
The liquid composition may be aqueous, such as an aqueous solution. The concentration
of the (one or more) bacteriocin(s) in said aqueous dispersion or solution may be
from 0.0001 to 10 mg/ml, preferably from 0.001 to 5.0 mg/ml, more preferably from
0.005 to 1.0 mg/ml, and most preferably from 0.01 to 0.2 mg/ml. If more than one bacteriocin
capable of exerting a cytotoxic effect on
Pseudomonas is employed, these concentrations relate to the total concentration of all such bacteriocins
of the invention.
[0041] The aqueous solution as said composition may, apart from the one or more bacteriocin,
contain a buffer. The buffer may be an inorganic or organic acid and/or salts thereof.
An example of an inorganic acid is phosphoric acid or salts thereof. Examples of the
organic acid are HEPES, acetic acid, succinic acid, tartaric acid, malic acid, benzoic
acid, cinnamic acid, glycolic acid, lactic acid, citric acid, and ascorbic acid. Preferred
organic acids are malic acid, lactic acid, citric acid, and ascorbic acid. The pH
of the solution may generally be from 6 to 8, preferably from 6.5 to 7.5. The pH of
the solution containing the buffer may be adjusted using hydrochloric acid or sodium
hydroxide solution.
[0042] Further, the solution may contain isotonic agents such as glycerol, mannitol, sorbitol,
a salt, or a combination of two or more such compounds. The salt is preferably an
alkali metal salt. A preferred salt to be used is sodium chloride. The aqueous solution
containing the one or more bacteriocin may be a buffered aqueous solution that may
contain further solutes e.g. salts such as from 50 to 400 mM NaCl, preferably from
100 to 200 mM NaCl. The aqueous solution may further contain a sulfhydryl compound
such as dithiothreitol (DTT), dithioerythritol, thioethanol or glutathione, preferably
DTT. The concentration of the total of sulfhydryl compounds in the aqueous solution
may be from 1 to 50 mM, preferably from 2 to 20 mM and more preferably from 4 to 10
mM.
[0043] Examples of carriers of the composition are solvents such as water or an aqueous
buffer (as described above); salts; sugars such as monosaccharides and disaccharides;
sugar alcohols; hydrophilic polymers such as polysaccharides, polyethylene glycol,
or hyaluronic acid (or its sodium salt); and other carriers known from pharmaceutical
compositions. Examples of polysaccharides are starch, cellulose and cellulose derivatives
such as hydroxypropyl cellulose, hydroxypropyl methylcellulose (HPMC), and carmellose.
Examples of sugars are glucose, fructose, lactose, sucrose, and maltose.
[0044] If the composition of the invention is a solid composition, it may be a powder such
as a lyophilized solid composition obtained by lyophilization of the extract or solution
mentioned above. The powder may contain additional solid components such as those
mentioned above for the aqueous solution. Before use, it may be reconstituted with
a suitable liquid, such as water or buffer. The solid composition may contain buffer,
salts or other components as mentioned above, such that the concentrations given above
may be achieved upon reconstitution or dissolution of the solid composition.
[0045] In a preferred embodiment, the composition is a pharmaceutical composition that comprises
one or more pharmaceutically acceptable carrier(s) or excipient(s). Examples of possible
carrier(s) and excipients were given above. A preferred pharmaceutical composition
is a liquid aqueous solution of the one or more bacteriocins. In another embodiment,
the composition is a solid formulation suitable for administration as an aerosol to
a patient.
[0046] For the treatment of keratitis, the pharmaceutical composition may be an ophthalmic
composition comprising a bacteriocin according to the invention. The ophthalmic composition
may be a sterile buffered solution of pH 7.0 to 7.5 having an osmolarity of 250-350
mOsm/L, preferably 270-330 mOsm/L. Possible buffers, if used, for ophthalmic compositions
are phosphate, citrate, tris (trometamol), or sodium hydrogencarbonate; citrate is
preferred. In one embodiment, the buffer is phosphate-free, and phosphate-free citrate-buffered
ophthalmic compositions are preferred. The concentration of the one or more bacteriocin
may be from 0.1 to 10 mg/mL, preferably from 0.5 to 8 mg/mL, more preferably from
1.0 to 5 mg/mL. As a wetting agent, a polysaccharide as mentioned above or sodium
hyaluronate may be contained as well.
[0047] The bacteriocin and the composition of the invention may be used in therapy. They
may be used in the treatment or prevention of a bacterial infection, preferably of
a bacterial infection by
Pseudomonas, such as
P. aeruginosa. In one embodiment, the bacteriocin or the composition is used in the treatment of
bacterial lung infection or bacterial pneumonia, preferably of lung infection or pneumonia
caused by
Pseudomonas, such as
P. aeruginosa. In another embodiment, the bacteriocin or the composition is used in the treatment
of keratitis, preferably of bacterial keratitis caused by
Pseudomonas, such as
P. aeruginosa.
[0048] The invention also provides a method of treating infection with
Pseudomonas, such as
P. aeruginosa of a subject in need thereof, comprising administering to said subject one or more
bacteriocin as described above or a composition as described above. The administration
is preferably topical, e.g. to the eyes in the case of keratitis or to the lungs in
the case of lung infection. The subject may be a human being or a mammal such as a
farm animal. Humans are preferred subjects. Generally, a liquid or solid pharmaceutical
composition containing the bacteriocin(s) and optionally further components as described
above is prepared for administration to the mammal. Liquid compositions may be aqueous
solutions as described above. Solid compositions may be powder containing the at least
one bacteriocin(s) e.g. in freeze-dried form.
[0049] For the treatment of keratitis, the bacteriocin(s) or the pharmaceutical composition
is administered into an affected eye, preferably in the form of an ophthalmic composition
as described above. The solution may be dropwise instilled into an affected eye. The
solution may be administered one drop into an affected eye three times per day. The
duration of treatment may be from one day until the infection has been effectively
treated, which may be from 2 to 10 days.
[0050] For the treatment of lung infection, the bacteriocin or the pharmaceutical composition
is administered into the lungs of the subject in need thereof. The bacteriocin or
the pharmaceutical composition may be administered into the lungs as a solid or liquid
aerosol. Thus, the invention also provides a pulmonary formulation comprising the
bacteriocin of the invention or the composition according to the invention. For an
overview over topical lung delivery of protein therapeutics see e.g.
Bodier-Montagutelli et al., EXPERT OPINION ON DRUG DELIVERY 2018, VOL. 15, NO. 8,
729-736; doi.org/10.1080/17425247.2018.1503251. The formulation may be a dry powder for aerosolization or a liquid solution for
nebulization. The composition may be administered to a subject 1 to 3 times per day.
The duration of treatment may be from one day until the infection has been effectively
treated, which may be from 2 to 10 days. The dosage may be from 10 µg to 500 µg, preferably
from 50 to 200 µg of bacteriocin of the invention per administration for a human subject.
[0051] A bacteriocin according to the invention may be produced by known methods of protein
expression in a standard expression system. Co-expression of the immunity protein
of the bacteriocin may be beneficial for preventing a toxic effect of the bacteriocin
on the host organism. For producing the bacteriocin, a nucleotide sequence encoding
it may be expressed in a suitable host organism. Methods usable for producing and
purifying a protein of interest have been described in the prior art and any such
methods may be used. An
E. coli expression system as generally known in the art may, for example, be used. If a eukaryotic
expression system is used, one or more introns may be inserted in the coding sequence
of the bacteriocin to prevent toxicity on the bacterial organism used for cloning.
[0052] Particularly efficient expression methods are plant expression systems that are also
known in the prior art. Plant expression systems usable for expressing a bacteriocin
according to the invention are described in the Examples. A possible way of achieving
expression of a nucleotide sequence of interest in plants is the use of self-replicating
(viral) replicons containing the nucleotide sequence encoding the bacteriocin. The
coding sequence of the bacteriocin may be codon optimized for expression in plants
or in the particular plant used as expression host. Plant viral expression systems
have been described in many publications, such as in
WO2012019660,
WO2008028661,
WO2006003018,
WO2005071090,
WO2005049839,
WO2006012906,
WO02101006,
WO2007137788 or
WO02068664 and many more publications are cited in these documents. Various methods for introducing
a nucleic acid molecule, such as a DNA molecule, into a plant or plant part for transient
expression are known. Agrobacteria may be used for transfecting plants with the nucleic
acid molecule (vector) or nucleic acid construct e.g. by agroinfiltration or spraying
with agrobacterial suspensions. For references, see
WO 2012019660,
WO 2014187571, or
WO 2013149726.
[0053] In embodiments wherein strong expression of a bacteriocin as a protein of interest
is desired, a nucleic acid construct containing a nucleotide sequence encoding the
bacteriocin may encode a viral vector that can replicate in plant cells to form replicons
of the viral vector. In order to be replicating, the viral vector and the replicons
may contain an origin of replication that can be recognized by a nucleic acid polymerase
present in plant cells, such as by the viral polymerase expressed from the replicon.
In case of RNA viral vectors (referred to as "RNA replicons"), the replicons may be
formed by transcription under the control of a promoter active in plant cells, from
the DNA construct after the latter has been introduced into plant cell nuclei. In
case of DNA replicons, the replicons may be formed by recombination between two recombination
sites flanking the sequence encoding the viral replicon in the DNA construct, e.g.
as described in
WO00/17365 and
WO 99/22003. If the replicon is encoded by the DNA construct, RNA replicons are preferred. Use
of DNA and RNA viral vectors (DNA or RNA replicons) has been extensively described
in the literature over the years. Some examples are the following patent publications:
WO2008028661,
WO2007137788,
WO2006003018,
WO2005071090,
WO2005049839,
WO02097080,
WO02088369,
WO02068664. Examples of DNA viral vectors are those based on geminiviruses. For the present
invention, viral vectors or replicons based on plant RNA viruses, notably those based
on plus-sense single-stranded RNA viruses may be preferably used. Accordingly, the
viral replicon may be a plus-sense single-stranded RNA replicon. Examples of such
viral vectors are those based on tobacco mosaic virus (TMV) and potexvirus X (PVX).
"Based on" means that the viral vector uses the replication system such as the replicase
and/or other proteins involved in replication of these viruses. Potexvirus-based viral
vectors and expression systems are described in
EP2061890 or
WO2008/028661. The method described in reference 11 may be used.
[0054] The bacteriocin may be expressed in a multi-cellular plant or a part thereof, notably
a higher plant or parts thereof. Both monocot and dicot (crop) plants can be used.
Common plants usable for expressing the protein of interest include
Nicotiana benthamiana, Nicotiana tabacum, spinach,
Brassica campestris, B. juncea, beets
(Beta vulgaris), cress, arugula, mustard, strawberry,
Chenopodium capitatum, lettuce, sunflower, cucumber, chinese cabbage, cabbage, carrot, green onion, onion,
radish, lettuce, field peas, cauliflower, broccoli, burdock, turnip, tomato, eggplant,
squash, watermelon, prince melon, and melon. Preferred plants are spinach, chard,
beetroot, carrot, sugar beet,
Nicotiana tabacum, and
Nicotiana benthamiana. In one embodiment, plants are used that do not normally enter the human or animal
food chain such as
Nicotiana species such as
N. tabacum and
N. benthamiana.
[0055] Generally, the bacteriocin as a protein of interest is expressed in the cytosol of
cells of the plants or plant parts. In this case, no signal peptide directing the
protein of interest into a particular compartment is added to the protein. Alternatively,
the protein of interest can be expressed in or targeted into chloroplasts of the plants;
in the latter case, an N-terminal pre-sequence, generally referred to as plastid transit
peptide or chloroplast targeting peptide, is added to the N-terminal or C-terminal
end, preferably the N-terminal end, of the bacteriocin as the protein of interest.
The bacteriocin may be co-expressed together with an immunity protein.
[0056] In the process of producing a composition comprising at least one bacteriocin, a
bacteriocin may, in the first step, be expressed in a plant or cells of a plant, such
as an edible plant. In the next step, plant material containing expressed bacteriocin
from a plant having expressed the bacteriocin is harvested. Plant material may e.g.
be leaves, roots, tubers, or seeds, or a crushed, milled or comminuted product of
leaves, roots, tubers, or seeds. In step (iii), the bacteriocin is extracted from
the plant material using an aqueous buffer. This may include that the plant material
is homogenized and insoluble material may be removed by centrifugation or filtration.
Soluble components including the bacteriocin will be extracted into the aqueous buffer
to produce a bacteriocin solution in the aqueous buffer. The aqueous buffer may contain
an inorganic or organic acid or salts thereof and may have a pH as defined above for
the aqueous solution as a composition of the invention. Further, the aqueous buffer
may contain salt and/or a sulfhydryl compound as also described above for the aqueous
solution as a composition of the invention. If a relatively pure bacteriocin composition
is desired, the bacteriocin solution in the aqueous buffer may be further purified
by removing undesired components in step (iv) according to known methods of protein
purification. The bacteriocin may be purified as described in reference
11.
EXAMPLES
Reference Example 1: Bacterial strains and cultures
[0057] Unless otherwise stated,
P. aeruginosa strains were prepared by culturing in Lysogeny Broth (LB) medium (Roth) or Casamino
Acids (0.5 % Bacto
™ Casamino acids, 5.2 mM K
2HPO
4, 5 mM MgSO
4) medium (BD Bacto) at 37 °C under shaking conditions (200 rpm); overnight cultures
were prepared by inoculation from frozen stocks.
P. aeruginosa strains used in experiments are described in
Suppl. Table 1.
Suppl. Table 1. P. aeruginosa strains used in the invention.
P. aeruginosa strain |
Isolated from |
Source |
Boston 41501 (ATCC 27853) |
Blood culture |
LGC ATCC |
PAO1 (ATCC 15692, DSM 22644) |
Infected wound |
Leibniz Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH |
PA14 (DSM19882) |
|
Leibniz Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH |
Bu002 LMG24892 |
Infected wound |
The BCCM/LMG bacteria collection |
A19 LMG25088 |
Infected wound |
The BCCM/LMG bacteria collection |
Pr335 LMG24969 |
Hospital environment |
The BCCM/LMG bacteria collection |
EY76 |
Infected eye |
A. Vitkauskienė LSMU |
BL77 |
Blood |
A. Vitkauskienė LSMU |
UR78 |
Urinary tract infection |
A. Vitkauskienė LSMU |
BR79 |
Bronchus |
A. Vitkauskienė LSMU |
BI80 |
Biopsy |
A. Vitkauskienė LSMU |
HP1 |
Hospital pneumonia |
A. Vitkauskienė LSMU |
HP6 |
Hospital pneumonia |
A. Vitkauskienė LSMU |
HP7 |
Hospital pneumonia |
A. Vitkauskienė LSMU |
HP40 |
Hospital pneumonia |
A. Vitkauskienė LSMU |
HP41 |
Hospital pneumonia |
A. Vitkauskienė LSMU |
HP52 |
Hospital pneumonia |
A. Vitkauskienė LSMU |
HP75 |
Hospital pneumonia |
A. Vitkauskienė LSMU |
PA-103 |
Sputum of patient |
ATCC 29260 |
12-35708 |
Cystic fibrosis |
A. Vitkauskienė LSMU |
13-18499 |
Cystic fibrosis |
A. Vitkauskienė LSMU |
12-29165 |
Cystic fibrosis |
A. Vitkauskienė LSMU |
180 (ATCC 19660) |
Human septicemia |
LGC ATCC |
NCTC 13437 |
|
Public Health England Culture collections |
NCTC 13921 |
Human throat |
Public Health England Culture collections |
Example 1: Construction of chimeric pyocins
[0058] The open reading frames encoding for PmnH (
P. synxantha EIK72868), Pflu095 (
P. fluorescens WP_016979095), Pflu373 (
P. fluorescens WP_014717373), Pflu794 (
P. fluorescens WP_081041794), Pflu618 (
P. fluorescens WP_034155618) and Pput259 (
P. putida WP_098964259) optimized for expression in
N. benthamiana were synthetized by Thermofisher Scientific (USA). Pyocin S5 synthesis and expression
vector construction were described previously
11.
[0059] Chimeric proteins were constructed as follows: N-terminal end of pyocin S5 (coding
sequence of 1-310 aa containing receptor binding and translocation domains of this
pyocin) was amplified with sequence-specific primers flanked by
Bsal recognition sites
(Suppl. Table 2). Cytotoxic domains of all non-
P.
aeruginosa putative bacteriocins were amplified with sequence specific primers flanked with
Bsal recognition sites
(Suppl. Table 2). Each killing domain fragment was paired with S5 fragment and both fragments were
inserted in Bs
al digested pICH29912, assembled TMV-based MagnICON vector
39. Obtained plasmids were used to transform
A. tumefaciens GV3101. The sequences of chimeric pyocins are presented below.
[0060] Suppl Table 2. Primers used to amplify receptor-binding- and translocation domain of S5 and pore-forming
domains of PmnH, Pflu095, Pflu794, Pflu618 and Pput259. In
italics - Bsal site,
underlined - overlapping sequence obtained after amplification,
bold - sequence complementary to template (synthetic gene with plant-optimized codons).
SEQ ID NO: |
Primer |
Sequence |
27 |
S5 fwd |
AAAGGTCTCACATGTCCAATGATAAC |
28 |
S5 rev |
AAAGGTCTCATTCTGTTCTCCTTCTTGTAGTCT |
29 |
PmnH fwd |
AAAGGTCTCAAGAAAAGGAACTTACACCAGATGAGAA |
30 |
PmnH rev |
AAAGGTCTCAAAGCTTAATTCAAAATAAG |
31 |
Pflu095 fwd |
AAAGGTCTCAAGAAAAGGATAAGGTTAGGACTGAGG |
32 |
Pflu095 rev |
AAAGGTCTCAAAGCTTAAAGGCTGG |
33 |
Pflu794 fwd |
AAAGGTCTCAAGAAAAGGCTAGCGACCAGGCTAAC |
34 |
Pflu794 rev |
AAAGGTCTCAAAGCTTAGAAGCCGA |
35 |
Pflu618 fwd |
AAAGGTCTCAAGAAGCTGAAGCTGAGGCTAAGAGA |
36 |
Pflu618 rev |
AAAGGTCTCAAAGCTTACCTTTGCTC |
37 |
Pput259 fwd |
AAAGGTCTCAAGAAGCAGAGGCTGAGGCTAAGAG |
38 |
Pput259 rev |
AAAGGTCTCAAAGCTTAGTGAGCAG |
Chimeric pyocin expression in plants
[0061] N. benthamiana plants were grown in a growth chamber at 25 °C with a 16 h light and 8 h dark photoperiod.
Four-to-six-week-old plants were used for vacuum infiltration with recombinant
A. tumefaciens.
[0062] Agrobacterium strains were inoculated from frozen stocks in 4 ml LB medium containing 50 µg/ml
rifampicin and 50 µg/ml kanamycin and cultivated at 28 °C with shaking at 220 rpm.
Overnight cultures were diluted 1:1000 starting from OD
595=1.0 in tap water and supplemented with 0.05 % Silwet L77 (Kurt Obermeier).
Agrobacterium suspension was poured into a desiccator vessel, connected to a vacuum pump. The entire
leaf system of a plant was then submerged into the suspension. Agroinfiltration was
achieved by applying (till pressure of 200 mbar) and releasing vacuum through the
pump. Plant leaves were harvested 5-6 days post agroinfiltration.
Purification of plant-produced chimeric pyocins
[0063] Frozen leaf tissue was homogenized with chilled mortar and pestle in liquid nitrogen.
Prepared powder was mixed with cold extraction buffer (50 mM Tris, 5 mM sodium acetate,
pH 5.0) at a ratio of 1 g of plant material to 5 ml of buffer. The crude extract was
incubated at 20-25 °C for 15-20 min. Cell debris were removed by centrifugation at
3220 g, at 4 °C for 20 min. Pellets were discarded and the supernatant was filtered
through membrane filter (pore size 0.45 µm). Ammonium sulphate was added up to 0.60
M and pH of solution adjusted to 8.0. The formed precipitate was removed by centrifugation
at 3220 g, at 4 °C for 5 min. The supernatant was taken as total soluble protein and
applied for purification in two steps.
[0064] At the first purification step, the chromatography column was filled with Phenyl
Sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden) and pre-equilibrated
with cold buffer (50 mM Tris, 5 mM sodium acetate, 0.60 M (NH
4)
2SO
4, pH 8.0). Protein solution was loaded to column and the Phenyl Sepharose bounded
protein fraction was eluted by washing with elution buffer (50 mM Tris, 5 mM sodium
acetate, 0.30 M (NH
4)
2SO
4, pH 8.0). Collected protein fraction was loaded into the diafiltrating concentrator
(10 kDa) and centrifuged at 3220 g until the volume of protein solution decreased
10 fold. The concentrate was then diluted up to a primary volume with 50 mM Tris and
5 mM sodium acetate (pH 8.0). The procedure was repeated till conductivity decreased
below 5 mS/cm and afterwards the protein solution was subjected to the final purification
step using Q Sepharose FF resin (GEHealthcare Life Sciences, Uppsala, Sweden). Chromatography
media was pre-equilibrated with cold buffer (50 mM Tris, 5 mM Sodium acetate, pH 8.0).
Protein solution was loaded to column and Q Sepharose unbounded protein was collected
in flow-through fraction. Collected protein fraction was loaded into the diafiltrating
concentrator (10 kDa) and centrifuged at 3220 g until the volume of protein solution
decreased 10 folds. The concentrate was diluted up to a primary volume with phosphate-buffered
saline (PBS) buffer. The protein was freeze-dried for long time storage. Pyocin S5
was purified as previously described by
11.
Results
Identification of Pseudomonas putative pore-forming bacteriocin sequences
[0065] Putative pore-forming bacteriocins from genus
Pseudomonas have been retrieved from NCBI by BLAST search using as query pore-forming domain
of pyocin S5 (pfam01024). After the analysis of BLAST results we selected six putative
pore forming bacteriocins from different
Pseudomonas species: Pflu095 (
P.
fluorescens WP_016979095), Pflu373 (WP_014717373 from strain A506, Pflu794 (WP_081041794 from
strain ATCC 17400), Pflu618 (WP_034155618 from strain H16), Pput259 (WP_098964259
from
P. putida strain FDAARGOS_376) and PmnH from
P. synxantha strain BG33R.
[0066] Clustal W amino acid sequence alignment of pore-forming domains showed 32-49% of
identity with pyocin S5 (
Fig. 1A). The amino acid sequences of pfam 01024 domains of bacteriocins were subjected to
phylogenetic tree analysis along with pore-forming domains of some described pore-forming
colicins, klebicins and pyocin S5
(Fig. 1B). Two major phylogenetic groups could be distinguished: pore forming domains of Pflu794,
Pflu373 and Pflu095 are most related to pyocin S5 and belong to the group of colE1-like
proteins, and Pflu618 and Put259 are most related to PmnH and belong to colA-like
or coIN-like protein group
(Fig. 1B).
Construction and plant expression of chimeric pyocins
[0067] Pore-forming domains of six selected
Pseudomonas putative porins were used for the construction of the chimeric proteins. All chimeric
proteins contain identical N-terminal end of first 309 amino acid residues of pyocin
S5 including translocation, FptA binding and CPA binding domains
17. The S5 fragment was fused to the cytotoxic domain of the putative pore-forming bacteriocins.
All six chimeric proteins were successfully expressed in
Nicotiana benthamiana transient expression system and purified by two-step chromatography (
Fig. 2).
Example 2: Agar disk-diffusion assay and determination of MIC by agar dilution method
Agar disk-diffusion assay
[0068] Overnight
P. aeruginosa cultures grown in CAA medium were equalized till OD
595=1.0 in CAA and diluted 100x. Sterile cotton swab was briefly submerged in diluted
microbial suspension, removing the excess of liquid by pressing it against the container
wall. The swab was used for evenly streaking bacteria on plates containing growth
medium with CAA solid agar (1.5%). 6 mm diameter sterile Whatman discs were placed
on soft-agar and 0.3-30 µg of chimeric pyocins were spotted to paper disks. The plates
were incubated overnight at 37 °C and bacteriocin inhibition zones were observed.
Results
[0069] A panel of 25
P. aeruginosa strains (from culture collections and clinical isolates,
Suppl. Table 1) were subjected to genomic DNA extraction and PCR analysis using pyocin S5, pyocin
S5 immunity protein and FptA receptor-coding gene specific primers. All 25 strains
tested positive for the presence of fptA. Six strains (PA14, PAO1, HP6, HP7, ATCC
1960 and NCTC 13921) tested positive for amplification of both pyocin S5 and pyocin
S5 immunity protein coding genes (
Fig. 8).
[0070] Next, all 25
P. aeruginosa strains were subjected to agar disc-diffusion assay by spotting different amounts
of purified pyocin S5 (0.3 µg, 3 µg and 30 µg) on bacteria lawns. Six
P. aeruginosa strains were resistant to pyocin S5. Four of these six strains were pyocin S5 and
immunity protein-encoding strains - PA14, PAO1, ATCC 1960 and NCTC 13921. Two cystic
fibrosis isolates, 12-35708 and 12-29165 were also completely resistant to pyocin
S5, despite the absence of S5, or S5 immunity protein coding genes. Surprisingly,
pyocin S5 still had week inhibition effect on the lawns of two remaining S5 producer
strains, clinical isolates HP6 and HP7. Turbid inhibition zones were detected when
3 µg and 30 µg of S5 were spotted on the lawn of these strains (
Table 1,
Fig 3).
[0071] We analyzed the activity of S5 chimeras in agar disc diffusion assay. Two chimeric
proteins, S5-Pflu095 and S5-PmnH demonstrated broadened activity spectrum in comparison
to pyocin S5. Both chimeras formed inhibition zones on the lawns of all six S5-producing
strains. The inhibition zones on HP6 and HP7 lawns were significantly larger and clearer
than those formed by S5. These chimeric proteins also demonstrated activity similar
to S5, with only small variations, on all other remaining
P. aeruginosa strains (
Table 1, Fig. 3). Between the two proteins, S5-PmnH demonstrated slightly superior activity and was
selected for further experiments.
[0072] Chimeric bacteriocin S5-Pflu373 also demonstrated good activity profile, similar
to S5-Pflu095, with exception that it was less active on one of S5 producers, ATCC
19660. S5-Pflu628 and S5-Pput259 chimeras performed less well, and had weaker and
less broad activity in comparison to pyocin S5, while S5-Pflu794 demonstrated only
week activity on 6 tested strains (
Table 1,
Fig. 3).
Table 1. Activity of chimeric pyocins on panel of
P. aeruginosa strains as determined in agar disc-diffusion assay. * - activity detected only with
30 µg of protein, ** - activity detected with 3 µg of protein, *** - activity detected
with 0.3 µg of protein. Pyocin S5 killing and immunity protein genes-containing strains
are in bold.
|
Colicin E1-like |
|
Colicin A-like |
|
S5 |
S5-Pflu095 |
S5-Pflu373 |
S5-Pflu794 |
S5-PmnH |
S5-Pflu618 |
S5-Pput259 |
Boston |
*** |
*** |
*** |
* |
*** |
** |
*** |
PA14 |
- |
** |
** |
- |
*** |
- |
- |
PAO1 |
- |
*** |
*** |
- |
*** |
** |
** |
Bu002 |
*** |
*** |
*** |
- |
*** |
** |
** |
A19 |
*** |
*** |
*** |
*** |
*** |
*** |
*** |
Pr335 |
*** |
*** |
*** |
** |
*** |
** |
*** |
EY76 |
*** |
*** |
*** |
- |
*** |
- |
- |
BL77 |
*** |
** |
** |
- |
** |
* |
* |
UR78 |
*** |
*** |
*** |
- |
*** |
* |
* |
BR79 |
*** |
** |
** |
- |
** |
- |
* |
BI80 |
*** |
*** |
*** |
- |
*** |
* |
* |
HP1 |
*** |
*** |
*** |
- |
*** |
** |
*** |
HP6 |
* |
*** |
*** |
* |
*** |
* |
** |
HP7 |
** |
*** |
*** |
- |
*** |
** |
** |
HP40 |
*** |
*** |
*** |
* |
*** |
** |
** |
HP41 |
*** |
*** |
*** |
- |
*** |
** |
** |
H P52 |
*** |
** |
** |
- |
** |
* |
* |
HP75 |
** |
* |
** |
- |
** |
- |
- |
ATCC 29260 |
*** |
*** |
*** |
- |
*** |
* |
* |
12-35708 |
- |
- |
- |
- |
- |
- |
- |
13-18499 |
*** |
** |
** |
- |
** |
* |
* |
12-29165 |
- |
- |
- |
- |
|
- |
- |
ATCC 19660 |
- |
* |
- |
- |
** |
- |
* |
NCTC 13437 |
** |
* |
* |
- |
* |
- |
- |
NCTC 13921 |
- |
*** |
** |
* |
*** |
* |
* |
Determination of MIC by agar dilution method
[0073] Aliquots of melted CAA (1.5% agar) medium (final volume - 25 ml) pre-wormed to 51
°C were supplemented with 0.1 mg/ml BSA and with appropriate amounts of pyocin stock
solutions of different concentrations (concentrations lowered by a factor of 2). Aliquots
were poured into Petri plates.
P. aeruginosa cultures were grown from single colony in CAA medium at 37 °C, 200 rpm until OD
595=0.2 and diluted to 10
7 CFU/ml in CAA medium. 1 µL of bacteria suspension were applied in three replicates
on each test plate. Plates were left to incubate in 37 °C overnight. The determined
MIC is a concentration of pyocin, where no confluent bacterial growth is observed.
Example 3: Ex vivo porcine corneas as model for bacterial keratitis
[0074] Preparation of porcine corneas. Porcine eyes were acquired from the nearest slaughterhouse. Enucleated eyes were
stored at -70 °C. Before the start of experiment, eyes were transferred to 4 °C for
1 hour, then to room temperature for 1-2 hours until the eyeballs were completely
defrosted. Then eyeballs were individually placed in sterile plastic containers and
submerged for 5 min in 2.5% Povidone-iodine (Betadine 100 mg/ml; EGIS Pharmaceuticals
PLC), then twice washed with sterile PBS. The corneas were excised with sterile surgical
blade No. #12. The excised corneas were stored in Minimum Essential Medium (MEM) supplemented
with Non-Essential Amino Acids, L-Glutamine (2 mM), penicillin (200 U/ml) + spectinomycin
(25 µg/ml) until further use at 4 °C up to two weeks.
[0075] Cornea infection and pyocin treatment. P. aeruginosa was grown overnight from frozen stock. Next morning, the culture was diluted 100
fold by fresh LB medium, and grown until OD595=~0.6 (~6 hours). Bacteria were collected
by centrifugation and resuspended in PBS. The dissected corneas were placed on agarose-gelatin
(0.5% each) solid support in a 6-well culture plate containing 800 µL MEM with antibiotics
(100 U/ml penicillin, 25 µg/ml spectinomycin), then three horizontal and 3 vertical
scratches were made using a sterile 25 gauge needle. 3×10
4 CFU of
P. aeruginosa ATCC 19660 strain or 0.4×10
4 CFU of PAO1 strain were applied to cornea and incubated for 16-20 hours in 37 °C
CO
2 incubator (20% CO
2). 5 corneas were used for each experimental point. After incubation with
P. aeruginosa, the corneas were visually inspected for opacity. The corneas which were clear and
without signs of infection were considered as non-infected and discarded from further
study. 5 µg of S5-PmnH (in 5 µl of PBS) or 5µl of PBS were applied to infected corneas
and incubated for additional 16-20 hours in 37 °C CO
2 incubator.
[0076] Homogenization and CFU counting. Prior the homogenization the corneas were washed 3 times for 10 min in 50 ml sterile
PBS with occasional agitation. Each cornea was chopped in 4 equal parts, trying to
get rid of sclera. 2 parts were placed in Precellys 24 tissue homogenizer (Bertin
technologies) tubes CKMix50-7ml and 2 ml PBS was added. Homogenization was performed
at following conditions: 6500 rpm 20 sec., 5 cycles, 3-5 min breaks on ice between
the cycles. The obtained homogenate was transferred into 15 ml Falcon tube and briefly
spun to sediment the big debris, then the supernatant recovered into new tube and
centrifuged at full speed for 10 min to pellet all bacteria. Bacteria were resuspended
in 200 µl sterile PBS, serially diluted and plated on LB-agar plates.
Results
[0077] Two types of
P. aeruginosa strains can be isolated from keratitis cases, cytotoxic
P. aeruginosa strains mainly cause keratitis related to contact-lens wear, while invasive strains
mostly cause disease in post-surgical complications
18. We aimed to investigate if both types of strains could be targeted by S5-PmnH in
disease models. Cytotoxic and invasive strains can be distinguished by genotyping
the effector protein coding genes of their type III secretion systems (TTSS). Invasive
strains were found to possess both exoS and exoT genes, whereas cytotoxic strains
appeared to have lost exoS but presented exoT and exoU genes
19-21. We selected for our experiments cytotoxic strain ATCC 19660 (exoT, exoU) and invasive
strain PAO1 (ExoY, exoT, exoS)
22. Both strains are pyocin S5 producers and immune to pyocin S5 but both are sensitive
to S5-PmnH.
S5-PmnH treatment can reduce P. aeruginosa bacterial numbers in porcine corneas ex
vivo
[0078] We first investigated the possibility to use S5-PmnH for eradication of
P. aeruginosa colonizing the cornea in an ex
vivo model, the dissected porcine corneas. Porcine corneas were colonized with invasive
strain PAO1 or cytotoxic strain ATCC 19660. S5-PmnH MIC determined by agar dilution
method against PAO1 was 4 µg/ml and against ATCC 19660 was 32 µg/ml.
[0079] In order to obtain
P. aeruginosa colonization, porcine corneas were incubated with 3×10
4 CFU of
P. aeruginosa ATCC 19660 or 0.4×10
4 CFU of
P. aeruginosa PAO1 for 16 to 20 hours. Then, 5 µg of S5-PmnH were applied to the cornea and incubated
for additional 16-20 hours. At the end of the experiment PAO1 burden in untreated
corneas reached an average of 7.6 log
10 CFU/cornea, while S5-PmnH-treated corneas the burden was only an average of 10 CFU
per cornea, demonstrating 6.6 log
10 reduction. In ATCC 19660 colonized corneas, S5-PmnH treatment reduced CFU number
by 5.3 log
10 (
Fig. 4). Thus, in ex vivo porcine corneas, S5-PmnH efficiently reduced
P. aeruginosa colonization by both strains.
Example 4: Murine keratitis model
Animals
[0080] The inbred mice of C57BL/6 strain of both sexes in equivalent numbers were used for
the research. 2-6 months old, adult female and adult male mice were purchased from
the Vilnius University vivarium of laboratory animals. The animals throughout the
period of the experiment were given standard chow and drinking water
ad libitum. Animals were housed in the individual plastic cages in a 12 h light/dark cycle at
21-23 °C. All regulated procedures on living animals were approved by The Lithuanian
Ethics Committee of Biomedical Research (Protocol no. B1-442) and were carried out
in accordance with the European Union legislation of OECD (directive 2010/63/EU).
The experiments were carried out in Biological research center of Lithuanian University
of Health Sciences.
P. aeruginosa keratitis induction and treatment
[0081] For
P. aeruginosa infection and keratitis induction the mice were anesthetized by Ketamine and Xylazine
90:9 mg/kg intraperitoneal injection. The cornea of the left eye of each mouse was
visualized under a stereoscopic microscope, and three 1 mm scratches were made using
a sterile 25 gauge needle. A 10 µL aliquot containing 4×10
6 cells of
P. aeruginosa ATCC 19660 (cytotoxic strain) or PAO1 ATCC 15692 (invasive strain) was applied to
the corneal surface. Depending on the experiment, the treatment was started 30 min
or 6 h post infection. 10 µl aliquot containing 0.14 mg of tobramycin or 20 µg of
S5-PmnH (both containing 0.5% hydroxypropyl methylcellulose (HPMC)) or PBS containing
0.5% HPMC was applied as one drop of substance to the each eye. HPMC is used as artificial
tears and was used in order to thicken the tears film and prolong the presence of
the applied product on the surface of the cornea. The treatment was continued for
five days twice daily. Mice were euthanized by cervical dislocation 1, 3, 5 days post
infection, and the eyeballs were collected and homogenized for viable bacteria count.
Assessment of clinical score
[0082] The eyes were examined and photographed with a dissection microscope equipped with
a digital camera at 1, 3 and 5 dpi to monitor the disease progression. At 1, 3 and
5 dpi disease severity was visually graded by using an established corneal damage
scale: 0, the pupil was partially or fully covered by clear or slight opacity; +1,
the anterior segment was partially or fully covered by slight opacity; +2, the pupil
was partially or fully covered by dense opacity; +3, the entire anterior segment was
covered by dense opacity; and +4, corneal perforation
40.
Histopathology
[0083] Histopathology experiments were carried out at Lithuanian University of Health Sciences,
Department of Veterinary Pathobiology of Veterinary Academy. One randomly chosen mice
of each study group was used for histopathology experiments. Enucleated eyes were
preserved in 10% formaldehyde. Eyes were paraffin-embedded, cut into 3-µm-thick sections,
deparaffinised, rehydrated and used for preparation of hematoxylin/eosin stained samples.
All samples were observed with the Eclipse TE2000-U microscope (Nikon, Tokyo, Japan).
Results
S5-PmnH efficiently kills bacteria and prevents acute disease in murine keratitis
models Infection by cytotoxic strain ATCC 19660
[0084] For induction of keratitis, the mice were anaesthetized, the cornea of left eye was
scratched with a sterile needle and
P. aeruginosa ATCC 19660 (4×10
6 CFU) was applied to the corneal surface. The treatment by S5-PmnH, tobramycin or
PBS (mock-treatment) was started 30 minutes post infection or 6 h post infection.
When the treatment was started 30 min after the infection, no viable
P. aeruginosa were isolated from infected eyes in both S5-PmnH and tobramycin-treated groups on
1, 3 or 5 dpi. By contrast, the bacterial burden in infected and untreated eyes reached
6-7 log
10 CFU/cornea (
Fig. 5A, left panel).
[0085] The visual inspection by microscope of infected untreated eyes revealed acute disease
signs: slight to dense opacity of cornea at 1 dpi, and dense opacity and sometimes
cornea perforation at 3 dpi. At 5 dpi all the mice had cornea perforations. No signs
of disease were observed in majority of samples treated by S5-PmnH or tobramycin (
Fig. 5A, right panel). The histopathology examination of infected and mock-treated eyes revealed
strong corneal inflammation at 1 dpi, and disrupted structure of the eye at 3 dpi
and 5 dpi. Only weak oedema in the corneal stroma was observed in all infected eyes
treated with PyoS5-PmnH and with tobramycin (
Fig. 5C.). Thus, both S5-PmnH and tobramycin completely eradicated
P.
aeruginosa ATCC 19660 and prevented the disease when the treatment was started almost immediately,
30 min, after infection.
[0086] We repeated the experiment with delayed treatment time, allowing the infection to
establish for 6 hours. Similar to the previous experiment, the average CFU burden
in infected mock-treated eyes reached 6.1 to 6.6 log
10 CFU/cornea. In the tobramycin-treated group of mice, no viable bacteria were isolated
on1, 3 or 5 dpi. In the S5-PmnH-treated group viable bacteria were isolated from one
mouse at 1 dpi (3.6 log
10 CFU/cornea) and from one mouse at 3 dpi (5 log
10 CFU/cornea). No viable bacteria were isolated from neither of three mice at 5 dpi
(
Fig. 5B, left panel). The clinical score evaluation of infected and mock-treated eyes revealed
very similar picture to the previous experiment: the opacity of cornea started at
1 dpi and 3 out of 4 mice had cornea perforations at 5 dpi. Only mild disease signs
were observed in two S5-PmnH-treated and one tobramycin-treated mice at 1 and 3 dpi
and all corneas were completely clear at 5dpi (
Fig. 5B, right panel). The histopathology examination of mock-treated eyes revealed the thinning
of corneal epithelium, thickening of stroma, acute inflammation at 1 dpi and acute
suppurated inflammation of cornea at 3 dpi and 5 dpi. PyoS5-PmnH-treated infected
eyes at 1 dpi presented signs of acute inflammation of cornea and no marked aberrations
at 3 and 5 dpi, just weak oedema of corneal stroma was observed. Tobramycin-treated
infected eyes at 3 and 5 dpi showed slight thickening of epithelium (
Fig. 5C, right panel). Thus, here again, S5-PmnH treatment was efficient in eradicating
P.
aeruginosa from infected corneas and in preventing the establishment and progress of disease.
Infection by invasive strain PAO1
[0087] We examined the efficacy of S5-PmnH for treatment of cornea infection by invasive
P. aeruginosa PAO1. The infection and treatment procedures were similar to the previous experiment
and treatment was started 6 h post infection. The control group of infected mice suffered
from severe disease and were euthanized at 3 dpi. At 1 dpi, bacterial burden in mock-treated
group of mice reached 6.26 log10 CFU/cornea. The S5-PmnH and tobramycin treatment
reduced burden by average 1.04 log10 CFU/cornea and 1.42 log10 CFU/cornea, respectively.
At 3 dpi, no viable bacteria were isolated from all three tobramycin-treated mice,
and from two S5-PmnH-treated mice. The cornea from the third S5-PmnH-treated mouse
contained 2.38 log10 CFU of viable bacteria. By contrast, burden in the control group
of mice reached 6.73 log10 CFU/cornea. At 5 dpi, no bacteria were isolated from two
tobramycin-treated and one S5-PmnH-treated mice. One mouse from the tobramycin treated
group contained 2.8 log10 CFU/cornea. The two remaining mice from the S5-PmnH treated
group contained 5.14 log10 and 3.34 log10 CFU/cornea (
Fig. 6A, left panel).
[0088] Clinical examination revealed stronger disease symptoms compared to the cytotoxic
strain ATCC 19660. Mock-treated eyes presented severe disease signs at 1 dpi (clinical
score 2-3). Several S5-PmnH-treated and tobramycin-treated eyes presented mild clinical
scores (1-2) starting from 1 dpi to the end of experiment (
Fig. 6A, right panel).
[0089] Histological examination of uninfected eyes revealed no marked aberrations, although
weak edema in corneal stroma was observed in most samples. Infected S5-PmnH-treated
eyes presented signs of acute inflammation of cornea at 1 dpi and 3 dpi. Infected
tobramycin-treated eyes presented signs of acute inflammation of cornea at 1 dpi,
thinning of cornea epithelium and strong edema of the corneal stroma at 3 dpi and
local inflammation of cornea, thinning and degeneration of cornea epithelium at day
5 (
Fig. 6C).
[0090] In conclusion, S5-PmnH efficiently reduced bacterial burden and prevented acute disease
regardless whether a cytotoxic or invasive strain was used for infection. However,
despite of lower S5-PmnH MIC against
P. aeruginosa PAO1 than against
P. aeruginosa ATCC 19660, the chimeric pyocin more efficiently eradicated the cytotoxic strain
of
P. aeruginosa and prevented the establishment of disease; a similar effect was observed for tobramycin.
Example 5: Murine lung colonization and treatment
Animals
[0091] CD1 male mice were supplied by Charles River (Margate, UK) and were specific pathogen
free. Mice were 11 to 15 g on receipt at the facility and were allowed to acclimatize
for at least 7 days.
[0092] Mice were housed in individual ventilated cages exposing the mice at all times to
HEPA filtered air. Mice had free access to food and water and were provided with aspen
chip bedding.
[0093] The room temperature was 22 ± 1°C, with a relative humidity of 60% and maximum background
noise of 56 dB. Mice were exposed to 12 hour light/dark cycles.
Mice infection and pyocin treatment
[0094] P. aeruginosa ATCC 27853 was recovered from long-term storage (-80°C) and cultured on cystine-lactose-electrolyte-deficient
(CLED) agar at 37 °C under aerobic conditions for approximately 16-24 h. 20 ml of
Mueller Hinton broth was inoculated with a single well isolated colony and cultured
overnight at 37 °C with shaking at 300 rpm65. The overnight broth was diluted 1:100
in Mueller Hinton broth and 100 ml was cultured in a baffle flask for ~6h at 37 °C
with shaking at 300 rpm until the broth OD was ~0.6 (~mid-exponential phase). 20 ml
of culture was centrifuged at 2465g for 10 minutes and washed in PBS. The pellet was
suspended in PBS and the OD
600 adjusted to 0.67 (~2.6×10
8 CFU/ml). The study inoculum was prepared from this by appropriate dilution with PBS.
The inoculum concentration was confirmed by quantitative culture on
Pseudomonas Selective Agar (PSA). The inoculum concentration for the study was 1.47 ×10
7 CFU/ml (5.87 ×10
5 CFU/mouse).
[0095] Mice were infected under temporary inhaled 3% isoflurane anesthesia by intranasal
(IN) instillation with 40 µl of the inoculum suspension split as 20 µl/nostril.
[0096] 1 hour post infection 6 mice were sacrificed in order to evaluate the pre-treatment
burden in lungs. Treatments were administered IN once at 1h post infection (50 µl
split to 25 µl/nares). Three treatment groups consisting of six mice each were administered
S5-PmnH (2.5, 25 and 250 µg). The fourth group of mice (n=6) was administered 200
µg Tobramycin (40 mg/ml injection solution, Hospira UK Ltd, diluted 10 times). The
study was terminated at 5 hours post infection for all animals. The clinical conditions
and weights were assessed and animals were immediately euthanized using an overdose
of pentobarbitone. Following confirmation of death, the lungs were excised and homogenized
in ice cold sterile phosphate buffered saline using a Precellys bead beater. The homogenates
were quantitatively cultured onto PSA agar and incubated at 37°C for 24 hours before
colonies were counted.
Results
S5-PmnH efficiently eradicates lung colonization by P. aeruginosa in a murine model of disease
[0097] Mice were infected intranasally (IN) with
P. aeruginosa ATCC 27853 strain. One hour later, 2.5, 25 and 250 µg/mouse of S5-PmnH was administered
IN once to both nares of the mouse. 5 hours later mice were euthanized and lung burden
of
P. aeruginosa ATCC 27853 was evaluated.
[0098] 5 h post infection,
P. aeruginosa ATCC 27853 burden in the mock-treated mice reached 1.24×10
7 CFU/g of lung tissue, corresponding to an increase of 1.53 log
10 CFU/g from 1 h post infection. S5-PmnH administered IN at 2.5 µg/mouse reduced lung
burden by 2.1 log
10 CFU/g, administered at 25 µg/mouse by 2.31 log
10 CFU/g and administered at 250 µg/mouse by 2.66 log
10 CFU/g. The bacterial burden was reduced to below the level of stasis (pre-treatment)
in all S5-PmnH treatment groups: in 2.5 µg/mouse group by 0.58 log
10 CFU/g, in 25 µg/mouse group by 0.78 log
10 CFU/g and in 250 µg/mouse group by 1.13 log
10 CFU/g. Increased reduction in burden was observed with higher dose levels of S5-PmnH,
however the differences were not statistically significant. Tobramycin administered
IN once at 200 µg/mouse reduced bacterial burden by 2.75 log
10 CFU/g compared to vehicle, corresponding to 1.23 log
10 CFU/g below the level of stasis. Higher variability was observed in this group compared
to S5-PmnH or vehicle treatments (Fig. 7).
General discussion of results
[0099] The attempts to use naturally occurring antibacterial proteins for fighting pathogenic
bacteria started a while ago. Bacteriophage lysins (endolysins), peptidoglycan hydrolases
were extensively exploited in this regard and have been shown to effectively target
numerous Gram-positive pathogens
23, the most advanced of those, lysin PlySs2 (Exebacase), has already entered Phase
III clinical trials. Similar to colicin-like bacteriocins, the modular structure of
bacteriophage lysins provides an opportunity to engineer enzymes with altered bacteriolytic
activity, and several active new hybrid molecules were constructed by swapping the
domains of different lysins
24. Fighting Gram-negative pathogens, however, is a much greater challenge as outer
membrane of the Gram-negative pathogens prevents the access of the potentially antibacterial
biologics to the periplasm or bacterial cell cytoplasm and nucleus. Several attempts
have been made to enable endolysins to penetrate the outer membrane. For instance,
endolysins have been engineered by adding polycathionic or/and hydrophobic/amphipathic
peptides to make them able to cross outer membrane (Artilysins)
25. As an alternative approach, the attempts have been made to equip colicin-like bacteriocins
with lytic domains of endolysins, thus enabling such engineered proteins to translocate
to the periplasm of Gram-negative bacteria
26,27. In first such attempt, the FyuA binding domain of pesticin, bacteriocin from
Yersinia pestis, was fused to the N-terminus of T4 lysozyme. This hybrid toxin was shown to kill specific
Yersinia and pathogenic
E. coli strains and, importantly, it was able to evade the pesticin immunity protein (Pim)
giving it a distinct advantage over pesticin
26. In another study,
P. aeruginosa bacteriocin pyocin S2 domains responsible for surface receptor binding and outer
membrane translocation were fused to the GN4 lysin to generate the PyS2-GN4 lysocin.
PyS2-GN4 induced peptidoglycan cleavage and log-fold killing of
P. aeruginosa, efficiently disrupted biofilms, and protected mice from
P. aeruginosa challenge in a bacteremia model
27. However, practical use of this chimeric bacteriocin is limited because of the narrow
activity spectrum as it only targets
P. aeruginosa strains carrying ferripyoverdine receptor FpvAI. These and other previous attempts
to engineer new active molecules for control of Gram-negative bacteria were invariably
relying on combining domains of phylogenetically and/or functionally unrelated proteins.
We therefore decided to construct chimeric molecules by swapping functional domains
of closely related bacterial species with the same mechanism of antibacterial activity,
namely chimeras of porin-porin type.
[0100] The only known pore forming bacteriocin of
P. aeruginosa was first detected in PAO1 strain
28. Pyocin S5 binds the highly conserved ferripyochelin FptA receptor
29. The exact prevalence of clinical
P. aeruginosa strains producing S5 is unknown, but it was demonstrated in the bacteriocin prevalence
study of catheter
P. aeruginosa isolates that about 25% of these strains contained pyocin S5 coding gene
30. Thus, these 25% of catheter isolates should be in theory resistant to pyocin S5.
We have tested pyocin S5 and S5 immunity protein gene presence in 25
P. aeruginosa isolates in our in house collection and found very similar results, 6 strains (or
24%) contained pyocin S5 and immunity protein coding gene sequences. As expected,
the presence of pyocin S5 coding gene in these strains correlated with the resistance
to the antimicrobial activity of pyocin S5. In our previous study, we also found that
pyocin S5 was active against 40% of tested
P. aeruginosa clinical isolates
11. Thus, we speculated that if the immunity of S5 producing strains could be overcome
by using cytotoxic domain other than S5, the spectrum of the chimera would be significantly
broadened, and it could be able to target more than half of strains.
[0101] Towards this goal, we constructed six chimeric S5 pyocins, in which the pore-forming
domain of S5 was replaced by pore-forming domains of putative bacteriocins from
Pseudomonas species other than
P. aeruginosa. All six chimeric pyocins were successfully expressed in
N. benthamiana transient expression system, which is our system of choice for expressing colicin-like
bacteriocins
11,31. Partially purified chimeric proteins were subjected to agar drop test assay on 25
P. aeruginosa strains, six between them being producers of S5 and insensitive or only slightly
sensitive to this porin.
[0102] The chimeric proteins demonstrated variable activity profiles, with three of them
standing out as more broadly active than the parental strain S5: two with bacteriocin-like
porin fragments coded by
P. fluorescens (S5-Pflu095 and, S5-Pflu373) and one with pore forming domain from
P. synxantha bacteriocin (S5-PmnH). PmnH has unusual architecture as it harbors two cytotoxic
domains, colicin-M like domain and also pore-forming domain. So far, only activity
of its pore-forming domain have been demonstrated
16. The chimeric protein S5-PmnH was active on all six S5-producing strains and did
show comparable activity to S5 on all remaining tested
P. aeruginosa isolates. This chimeric protein was selected for further experiments in murine models
for topical treatment of two unrelated models of disease caused by
P. aeruginosa: keratitis model and lung infection model.
[0103] Bacterial keratitis is mostly affecting contact lens wearers, although several other
risk factors, such as ocular surface diseases, ocular traumas or ocular surgery, are
also not negligible. A 5-year review of cases at Dubai hospital revealed that in 37%
of bacterial keratitis cases the causative agent of disease is
P. aeruginosa, although this frequency may vary in different geographical regions
32,33. Untreated bacterial keratitis usually results in blindness.
[0104] Two types of
P. aeruginosa strains are found in eyes affected by keratitis: cytotoxic strains that mainly cause
keratitis in contact-lens wearers, and invasive strains mostly causing disease in
post-surgical complications
18. Cytotoxic and invasive strains can be distinguished by genotyping the effector protein
coding genes of their type III secretion systems (TTSS). Invasive strains were found
to possess both exoS and exoT genes whereas cytotoxic strains appeared to have lost
exoS but presented exoT and exoU genes
19-21. In our experiments, we chose cytotoxic strain ATCC 19660 (exoT, exoU) and invasive
strain PAO1 (ExoY, exoT, exoS)
22. Both strains are pyocin S5 producers and are immune to pyocin S but were confirmed
to be sensitive to the chimeric pyocin S5-PmnH.
[0105] Chimeric pyocin S5-PmnH demonstrated efficacy in both cytotoxic and invasive
P. aeruginosa models of eye infection. In the cytotoxic keratitis model, the treatment completely
eliminated all bacteria one day post infection, if treatment was started 30 min after
infection. When treatment was delayed for 6 hours, viable bacteria were isolated from
one mouse at 1 dpi and one mouse at 3 dpi. Visual inspection and histological examination
of eyes of all treated mice in both experiments did not reveal any significant lesions,
with no marked difference from non-infected eyes. By contrast, very strong keratitis
symptoms were observed in all infected eyes at 3 and 5 dpi. Thus, S5-PmnH treatment
efficiently eradicated
P. aeruginosa from cornea infected by the cytotoxic strain
P. aeruginosa ATCC 19660 and prevented the establishment and progress of the disease.
[0106] A similar experiment was performed using the invasive
P. aeruginosa strain PAO1 with treatment starting 6 hours after infection. Although both treatments
(S5-PmnH or tobramycin) were less efficient in eradicating bacteria, the progress
of disease was greatly reduced in all treated mice. Taken together, these studies
clearly demonstrate the efficacy of chimeric pyocin in this disease model, comparable
to that of the standard of care antibiotic tobramycin.
[0107] P. aeruginosa is also a frequent cause of lung infections, including hospital-acquired pneumonia
(HAP) and ventilator-associated pneumonia (VAP). This pathogen has a worsening global
trend towards more likely displaying MDR phenotypes
34. In addition,
P.
aeruginosa can cause chronic lung infections in patients with cystic fibrosis (CF) and non-CF
bronchiectasis. Acquisition of
P.
aeruginosa is associated with increased morbidity and mortality in patients with CF, and is
an important factor in the development and progression of CF respiratory disease
35-37. Patients with CF are at very high risk of developing infections with multidrug-resistant
(MDR)
P. aeruginosa, owing to the frequent and often prolonged courses of oral, intravenous, and aerosolized
antibiotics that are used to treat the chronic lung disease of CF
38. In our validated mouse lung colonization model, one single application of chimeric
pyocin S5-PmnH, even at the lowest concentration used, 2.5 µg, reduced bacterial burden
in the lungs below the level of stasis. The successful results obtained with chimeric
pyocin S5-PmnH used in our study show that such bacteriocins can be used for efficiently
targeting bacteria in lungs and surprisingly demonstrate engineering bacteriocins
with modified or broadened activity spectra.
AMINO ACID AND NUCLEIC ACIID SEQUENCES
[0108]
SEQ ID NO: 1 Pyo S5 first segment
SEQ ID NO: 2: Pyo S5 first segment coding sequence
SEQ ID NO: 3: PmnH second segment
SEQ ID NO: 4: PmnH second segment coding sequence
SEQ ID NO: 5: S5-PmnH, pyocin S5 part is underlined.
SEQ ID NO: 6: S5-PmnH coding sequence, pyocin S5 part is underlined
SEQ ID NO: 7: Pflu095 second segment
SEQ ID NO: 8: Pflu095 second segment coding sequence
SEQ ID NO: 9: S5-Pflu095, pyocin S5 part is underlined
SEQ ID NO: 10: S5-Pflu095 coding sequence, pyocin S5 part is underlined
SEQ ID NO: 11: Pflu373 second segment
SEQ ID NO: 12: Pflu373 second segment coding sequence
SEQ ID NO: 13: S5-Pflu373, pyocin S5 part is underlined
SEQ ID NO: 14: S5-Pflu373 coding sequence, pyocin S5 part is underlined
SEQ ID NO: 15: Pflu794 second segment
SEQ ID NO: 16: Pflu794 second segment coding sequence
SEQ ID NO: 17: S5-Pflu794, pyocin S5 part is underlined
SEQ ID NO: 18: S5-Pflu794 coding sequence, pyocin S5 part is underlined
SEQ ID NO: 19: Pflu618 second segment
SEQ ID NO: 20: Pflu618 second segment coding sequence
SEQ ID NO: 21: S5-Pflu618, pyocin S5 part is underlined
SEQ ID NO: 22: S5-Pflu618 coding sequence, pyocin S5 part is underlined
SEQ ID NO: 23: Ppu259 second segment
SEQ ID NO: 24: Ppu259 second segment coding sequence
SEQ ID NO: 25: S5-Ppu259, pyocin S5 part is underlined
SEQ ID NO: 26: S5-Ppu259 coding sequence, pyocin S5 part is underlined
SEQ ID NOs: 27-38: primer sequences shown in Suppl. Table 2
SEQ ID NO: 39: Pyo S5 second segment (killing domain)
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